Platelet aggregation test and device

ABSTRACT

An assembly for testing platelet aggregation including an electrode subassembly that is mounted in a cuvette subassembly for use with relatively small samples containing platelets.

CROSS-REFERENCE TO RELATED APPLICATIONS

This continuation application claims the benefit under 35 U.S.C. §121 ofapplication Ser. No. 14/730,770 filed on Jun. 4, 2015, entitled PLATELETAGGREGATION TEST AND DEVICE, which is a continuation of application Ser.No. 13/998,758 filed on Dec. 3, 2013, which is a continuation ofapplication Ser. No. 13/275,402 filed on Oct. 18, 2011 and whose entiredisclosures are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to devices for measuring platelet aggregation.

BACKGROUND OF THE INVENTION

Platelets are a component of blood that can aggregate when necessary forwound healing, for example. However testing platelet aggregation canreveal signs of a bleeding disorder or risk of thrombosis, or if apatient is not responding to anti-platelet therapy. Platelet aggregationtests can help diagnose problems with platelet function and determinewhether the problem is due to one's genes, another disorder, or a sideeffect of medicine.

Platelets are known to aggregate under a variety of conditions and inthe presence of a number of different reagents. “Platelet aggregation”is a term used to denote the adherence of one platelet to another. Whenthey aggregate, platelets change from a discoid shape to a morespherical form, extend long processes known as pseudopodia and becomesticky. As a result, the platelets stick to one another and to thedamaged tissue, thus plugging gaps or holes in the blood vessel wall.Although the primary response of platelets is to aggregate, a secondaryrelease reaction may also occur, during which platelets releasematerials which accelerate the clotting process.

Platelets' ability or inability to respond to particular aggregatingreagents is the basis for differentiating platelet dysfunction fromnormal, for example.

Aggregation can be induced in a sample by adding aggregating agents toplatelet-rich plasma or whole blood. Platelet aggregation depends on thepresence of calcium, fibrinogen and one or more plasmatic factors, andan aggregating agent. Platelet aggregation will vary with differentaggregating agents and with their concentration.

One method of testing platelet aggregation is optical aggregometry. Forexample, in 1962, Born described the aggregation of platelets by ADP andmodified a colorimeter to monitor continuously this aggregation inplatelet rich plasma. These modifications included incubation at 37° C.,stirring and recording the change in light transmission over time on apen recorder. This method is commonly referred to as Light TransmissionAggregometry (LTA).

With LTA, platelets are in a suspension of plasma isolated from ananticoagulated blood sample by a relatively low centrifugal forcecentrifugation. This material is known as platelet-rich plasma (PRP).Platelet-poor plasma (PPP) is prepared by centrifuging the blood sampleat a relatively high force.

The sample chamber or chambers in multiple channel instruments areheated to 37° C. Provision is made for stirring of the sample becauseplatelet to platelet contact is necessary to the determination of invitro platelet aggregation. A beam of light shines through the samplecuvette. Photodiodes detect the light able to pass through the sample. Asample cuvette containing PRP is measured and a sample cuvettecontaining PPP is measured. PRP is arbitrarily considered to be 0% lighttransmission or 0% aggregation; PPP is considered to be 100% lighttransmission or 100% aggregation. The difference in light transmissionoutputs from the photodiodes is transferred to recording devices.

When an agonist or aggregating agent is added to the cuvette containingPRP and the platelets respond, changes in light transmission occur andare recorded over time by the recording device.

When the platelets undergo shape change in response to an agonist, theirlarger size allows less light to pass through the PRP: this is recordedas less light transmission through the sample relative to the PPP. Ifthe dose of aggregating agent is strong enough to cause the platelets toadhere to each other and form aggregates, more light is able to passthrough the PRP sample. The change in light transmission recorded, overtime, shows a trend towards the platelet poor plasma, or 100% lighttransmission.

As is well known, in-vitro aggregation recordings are characterized bytheir appearances:

-   -   shape change    -   a first wave of aggregation (primary aggregation) that may        reverse and return towards the PRP baseline    -   Irreversible second wave aggregation that occurs when the        platelets' secreted granule contents become the stimulus and        cause additional aggregation.

Aggregation curves are also characterized by:

-   -   the maximum amount of change in light transmission caused by the        agonist (percent aggregation)    -   the slope-or rate-of the aggregation, in % change of aggregation        per minute.

Multiple aggregating agents and dosages are usually used to stimulatethe platelets. Different aggregating agents stimulate different pathwaysof activation in the platelets: either binding sites or metabolicpathways. Different concentrations of agonists are used to elicit afamily of curves (dose response curves).

The pattern of responses to these test panels is compared to establishednormal response patterns and established abnormal response patterns.This information is considered to relate to the platelet functioncomponent of homeostasis.

In 1980, Cardinal and Flower described an impedance method for measuringaggregation in whole blood (U.S. Pat. No. 4,319,194). In their method, avery small electric current is passed between two electrodes. Duringinitial contact with the blood the electrodes become coated with amonolayer of platelets. When an agonist is added, platelets aggregate onthe monolayer increasing the impedance. This increase in impedance isrecorded on a pen recorder.

In the impedance method, platelets are tested in anti-coagulated blood,without the need to isolate them from other components of blood. Becausethere is no need to centrifuge the specimen to produce an opticallytransparent suspension of cells, the entire platelet population istested. The process of testing consumes less technical time; and labilefactors in the blood itself that may influence platelet function arepreserved.

A typical impedance aggregometer consist of a sample chamber or chambers(in multiple channel instruments,) heated to 37° C. The device typicallyincludes apparatus to stir the samples, commonly utilizing non-magneticdisposable stir bars. Cuvettes containing the test sample and a stir barare placed in the chamber(s).

The impedance (or electrical resistance) method of aggregation isnon-optical. An electrode probe assembly is inserted into a cuvettecontaining a test sample. The electrode probe assembly consistsbasically of two precious metal wires that are immersed in the sample.An AC voltage in the millivolt range is applied to the probe circuit.The instrument measures the electrical resistance or impedance betweenthe two immersed wires.

During a brief period of equilibration, a monolayer of platelets formson the exposed portions of the wires, resulting in a stable impedancevalue. This stable baseline of impedance is assigned a value of zeroohms of resistance. An agonist is added to the cuvette and thestimulated platelets aggregate to the platelet monolayer on the immersedwires. This accumulation of platelets adds electrical resistance to thecircuit. The changes in resistance are measured and quantified in ohms(the measurement of electrical resistance). The impedence measurement ofthe aggregated sample is generally run continuously for four to sixminutes after the addition of an agonist.

Results of impedance aggregation tests are quantified by:

-   -   Ohms of aggregation at a given time in the test    -   Slope, or rate of the reaction, in ohms change per minute    -   Maximum extent of aggregation, in ohms.

The increase in impedance is directly proportional to the mass of theplatelet aggregate. Impedance aggregation in blood is more sensitive tothe aggregating effects of ristocetin so it may be more sensitive to vonWillebrand disease than the bleeding time or vWF (ristocetin co-factor)assay. Impedance aggregation in blood is not dependent on the opticalcharacteristics of the sample, so tests can be performed on lipemic andthrombocytopenic samples. As centrifugation is not required, impedanceaggregation is especially useful in conditions where megathrombocytecount is increased.

The impedance method allows the study of platelets in the morephysiologically representative whole blood environment. Samplepreparation is greatly simplified, and preserves labile modulators suchas prostacyclin and thromboxane A2, resulting in a testing environmentproven to be more sensitive to the effects of many anti-platelet drugs(e.g., aspirin, dipyridamole, abciximab, clopidogrel, ticagrelor,ticlopidine, prasugrel, etc. . . . ).

In 1984, Freilich developed a low cost disposable electrode formeasuring impedance aggregation by substituting for the wire electrodesconductive ink printed on a plastic nonreactive base (U.S. Pat. No.4,591,793). This device is less expensive than the Cardinal device andis disposable after each test; however, there are disadvantages to theFreilich device. The platelets have difficulty adhering to the exposedconductive surface of the Freilich device, probably due to the surfacebeing thin. Sometimes the aggregated platelets break off the surface,causing a sudden change in impedance. Although the Freilich device isinexpensive to manufacture, the measurements returned by the device canbe inconsistent and not reproducible.

In 1997 Freilich et al. developed an improved low cost disposableelectrode that overcame the reproducibility problems of their priordevice (see, U.S. Pat. No. 6,004,818). The inventors discovered that themost reproducible configuration has the electrodes side-by-side withrespect to the flow pattern. This configuration allows the platelets tostick to the face and the area between the electrodes, facilitating theformation of a bridge of platelets between the electrodes, which resultsin a stronger bond of platelets to the electrodes.

This device consists of two metal plates with a connection tab at oneend and tip at the other end. The two plates are separated by anelectrical insulator comprised any non-conducting material, such asmylar, plastic or teflon, which will separate the electrodes by theproper amount. Except for the tips, the plates are isolated from thesample by a non-conductive coating comprised of any insulating material,such as plastic or epoxy, which is non-reactive with the blood sample.The electrode tips are side by side with respect to the flow pattern.The tips are non-circular in cross-section, preferably rectangular, andmost preferably square. The advantages of square tips are that at leastone planar face of one electrode tip is adjacent and parallel to atleast one planar face of the opposing electrode tip. Also, the squareelectrode tips are easier to produce than round electrode tips because astamping process can be used to make the electrode out of flat metal toform an electrode plate.

The position fixing means are either a pair of molded plastic,semi-circular fins extending outwardly from the molded plastic coatingthe electrodes or molded plastic parts with slots for the placement ofthe electrodes. It is of considerable importance to keep the electrodetips in the proper placement in the cuvette so it is necessary to havethese elaborate means to hold the electrode assembly in place.

There are several disadvantages to this device, which prevented it fromever going to market. First although it is less expensive than theCardinal and Flower device, it is still too expensive to be disposablein today's cost conscious laboratory. Additionally, this device isdifficult to manufacture which would result in a high rejection rate.Therefore this device has never been produced nor sold.

However, a variation of this device was produced and sold. This deviceused Square electrode pins held in the optimal position in the sample.The main difference is instead of plates the pins were attached to akapton strip that had electrical circuit lines on it These circuit lineselectrically connected the pins to tabs at the top of the a kaptonstrip. These tabs are used as contacts for connecting to the electricalcircuit of the instrument.

This design works well in a 1 mL sample, however, when it was adapted itto a smaller sample, there was a spontaneous reaction of the plateletsmost likely due to a sheer force in the smaller sample cuvette. Thisspontaneous reaction makes this device unusable in a small samplecuvette. The small sample size is more beneficial because less patientblood is needed to run the assay.

Because the prior design failed when it was adapted to fit a smallersample, there is a need for a workable electrode for smaller bloodsamples. This electrode needs to be easy to use and low cost and havethe ability to test platelet function in whole blood in a small samplesize.

The prior design was used in a 1 mL sample size, in a large cuvette(0.44″×1.83″). With the new device, the sample size will be from 250 μLto 300 μL. This smaller sample size requires a smaller sample cuvettewith provision to stir the sample at 1000 RPM, the traditional stirringspeed for platelet aggregation.

SUMMARY OF THE INVENTION

This invention is an assembly for measuring platelet aggregationcomprising an electrode subassembly and a cuvette subassembly.

The electrode subassembly comprises:

-   -   i. an electrically non-conductive substrate with two wings at        its top extending horizontally away from each other in opposite        directions with a downwardly extending member;    -   ii. a pair of electrode contact pads separated from each other        and mounted on the wings of the substrate, each contact pad        having an electrically conductive lead extending downwardly on        the downwardly extending member of the substrate, each lead        having electrically connected to it a conductive wire extending        below and downwardly away from the downwardly extending member,        each of the two wires having a horizontal cross-section that is        rounded with a cross-sectional dimension from about 0.17 to        about 0.38 mm, where the portions of the two wires that extend        downwardly away are substantially parallel, are spaced from one        another about 0.18 to about 0.42 mm apart and are from about 1.9        to about 4.5 mm long;

And the cuvette subassembly comprises:

-   -   i. a lower reservoir to receive a sample containing platelets,        the lower reservoir having a substantially flat, closed bottom        and having a substantially cylindrical wall, the lower reservoir        having an internal volume of from about 225 to about 375 μL, the        reservoir adapted to receive a sample containing platelets;    -   ii. extending upwardly from the lower reservoir, an upper body        comprising:        -   a. a pair of arms and a back member with a slot formed            between each arm and the back member, each slot adapted to            receive a wing from the electrode subassembly;        -   b. the dimensions of the cuvette subassembly being such that            when the wings of the electrode subassembly are positioned            in the slots, the two wires are positioned in the reservoir            and above the bottom of the reservoir and adapted to be            submerged in a sample placed in the reservoir;        -   c. a channel from an upper part of the cuvette subassembly            to approximately the upper part of the reservoir, adapted to            allow reagent to be placed into the sample without            substantially interfering mechanically with the electrode;

The assembly also can include a stir bar adapted to be placed in thebottom of the cuvette well and free to rotate under the influence of anoutside source to create a flow of sample in the well and between thetwo wires of the electrode subassembly when the electrode subassembly ismounted in the cuvette subassembly, the distances between bottom ends ofthe two wires and the bottom of the lower reservoir being such that thestir bar can fit in that distance and still create such flow.

Other aspects of the invention are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the electrode subassembly of thisinvention.

FIG. 2 is a front view of the electrode subassembly of this invention.

FIG. 3 is a side view of the electrode subassembly of this invention,viewed from the right side of FIG. 2.

FIG. 4 is a perspective view of the cuvette subassembly of thisinvention.

FIG. 5 is a top view of the cuvette subassembly of this invention.

FIG. 6 is a bottom view of the cuvette subassembly of this invention.

FIG. 7 is a cross-sectional view of the cuvette subassembly of thisinvention taken along the plane of line VII-VII of FIG. 6.

FIG. 8 is a rear view of the cuvette subassembly of this invention withthe electrode subassembly of this invention inserted into it.

FIG. 8A is a rear view, partially in shadow of the electrode subassemblyinserted into the cuvette subassembly of this invention with a stir barin place.

FIG. 9 is a perspective view of a heater block/stirrer assembly usedwith the cuvette and electrode subassemblies of this invention.

FIG. 10 is a perspective view of a heater block/stirrer assembly usedwith the cuvette and electrode subassemblies of this invention, withthose subassemblies placed into the heater/stirrer assembly.

FIG. 11 is a first end view of a heater block/stirrer assembly used withthe cuvette and electrode subassemblies of this invention, with thosesubassemblies placed into the heater/stirrer assembly.

FIG. 12 is a cross-sectional first end view of a heater block/stirrerassembly used with the cuvette and electrode subassemblies of thisinvention, with those subassemblies placed into the heater/stirrerassembly, taken along the plane of line XII-XII of FIG. 11.

FIG. 13 is a second end view of a heater block/stirrer assembly usedwith the cuvette and electrode subassemblies of this invention, withthose subassemblies placed into the heater/stirrer assembly. This secondend view is the opposite end from the first end view.

FIG. 14 is a cross-sectional second end view of a heater block/stirrerassembly used with the cuvette and electrode subassemblies of thisinvention, with those subassemblies placed into the heater/stirrerassembly, taken along the plane of line XIV-XIV of FIG. 13.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

This invention includes a disposable assembly (or kit) 10 (see FIG. 8,for example) for measuring platelet aggregation in a sample. The samplecan be whole blood or a blood component containing platelets. Forreference purposes, we use the terms “front,” “back,” “upper,” and“lower” to orient the reader and assist in his/her understanding of theinvention. We could have just as easily used terms such as “first” and“second” or “left” and “right” or “distal” and “proximal.” Our choice ofterms of orientation is not meant to limit the scope of this invention,if one wants, for example, to create a mirror image of this invention orrotate its elements in space.

The disposable assembly 10 for measuring platelet aggregation includesan electrode subassembly 12 (see FIGS. 1-3, 8, 10, 12 and 14) and acuvette subassembly 14 (see FIGS. 4-8, 10, 12 and 14). The disposableassembly is used with a heater/stirrer assembly 16 (see FIGS. 9-14).Disposable assembly also utilizes a stir bar 18 (FIGS. 8A and 12) thatwill be discussed further below.

Electrode subassembly comprises an electrically non-conductive substrate20 (FIGS. 1-3) with two wings (22 a and 22 b)(see also FIG. 8) at itstop extending horizontally away from each other in opposite directionswith a downwardly extending member 24.

On substrate 20 are pair of conductive electrode contact pads 26 a, 26 b(FIGS. 1-3, 8 and 12) separated from each other and mounted on the wingsof the substrate, each contact pad having an electrically conductivelead 28 a, 28 b mounted on and extending downwardly on the downwardlyextending member 24 of the substrate. By “mounted on” we mean any kindof mounting but preferably by electrodeposition. The leads 28 a and 28 bare preferably coated with a non-conductive material (e.g., polymeric)to avoid exposure of those leads to the test sample when the electrodesubassembly 12 is mounted in the cuvette subassembly with a sample inthe cuvette reservoir.

Each lead (or tracing) 28 a, 28 b has electrically connected to it aconductive wire 30 a, 30 b extending below and downwardly away from thedownwardly extending member 24. Each of the two wires having ahorizontal cross-section that is rounded with a cross-sectionaldimension from about 0.17 to about 0.38 mm. By “rounded” we mean oval,circular and the like as opposed to square or rectangular with sharpedges. The wires are preferably made from palladium (or its alloys), butother metals such as platinum, rhodium, gold, iridium, osmium, rheniumand ruthenium or alloys of the same can also be used.

As shown in FIGS. 1, 2 and 8A, the portions of the two wires 30 a and 30b that extend downwardly away are substantially parallel, are spacedfrom one another about 0.18 to about 0.42 mm apart and are from about1.9 to about 4.5 mm long (as measured from points d and e on FIGS. 2, 3and 8 a, those points identifying the portions of wires 30 a, 30 b thatare not overlaying substrate 20). As shown in FIGS. 1-3 and 8A, member24 has a lower, narrow protuberance 32 on which the upper ends of wires30 a and 30 b are mounted. The purpose of narrowing the lower part ofmember 24 to a protuberance 32 is ultimately to allow the sample, underinfluence of stirring as described below, to flow around the fluidreservoir of cuvette 14 as evenly as possible, and around and betweenthe wires during stirring.

To lend some optional structural support to substrate 20 andparticularly wings 22 a, 22 b, one can add a rigid or semi-rigidpolymeric reinforcing member 34 across the wings (FIGS. 1 and 3). Theadditional thickness and strength that member 34 provides affords abetter fit—preferably an interference fit—into tapered slots 38 a, 38 b(see e.g. FIGS. 4, 5 and 7) in cuvette subassembly 14 as describedbelow.

Finally, electrode subassembly 12 preferably includes an electricallynon-conductive spacer element 36 (FIGS. 1-3, and 8A) fixed to the lowerends of wires 30 a and 30 b to assist in holding them in a fixedparallel position to each other. Both the substrate 20 and the spacerelement 36 are preferably made from polyimide film, preferably Kaptonbrand.

The cuvette subassembly 14 of this invention (e.g., FIGS. 4, 7, 8 and8A) includes a lower reservoir 40 to receive a sample containingplatelets. The lower reservoir has a substantially flat, closed bottom42 and a substantially cylindrical wall 44. By “substantiallycylindrical,” we do not mean to exclude some tapering that isillustrated in the drawings. Lower reservoir 40 has an internal volumeof from about 225 to about 375 μL and the reservoir is adapted toreceive a sample containing platelets.

Extending upwardly from lower reservoir 40 is an upper body 46 (FIGS. 4and 7) that includes a pair of arms 48 a, 48 b. Upper body 46 also has aback member 50 that creates a slot 38 a, 38 b (preferably tapered)between each arm 48 a, 48 b and back member 50, each slot is adapted toreceive a wing 22 from the electrode subassembly (see FIGS. 4, 5, 7, and12). As mentioned above, slots 38 a, 38 b are tapered, being wider atthe top than the bottom 52 (FIGS. 7 and 12). As mentioned above, thewings 22 a, 22 b with reinforcing member 34 behind them create athickness which in this case is adapted to fit snugly into the bottom 52of each of the slots 38 a, 38 b. This preferred fitment holds theelectrode subassembly into the cuvette subassembly so that the two holdrelatively fixed positions relative to one another when assembled and inuse.

The dimensions of cuvette subassembly 14 are such that when the wings ofthe electrode subassembly 12 are positioned in slots 38 a, 38 b, the twowires 30 a and 30 b are positioned in reservoir 40 and above the insidebottom of reservoir 40 and adapted to be submerged completely in asample placed in reservoir 40. In addition, the dimensions of the twosubassemblies 12 and 14 are such that there is sufficient space betweenspacer element 36 and the inside bottom of reservoir to allow amagnetically drivable stir bar 18 to fit easily and without interferenceon the inside bottom of the reservoir (see, e.g., FIGS. 8A and 12).

In addition, as shown in FIG. 8A, on electrode subassembly 12, theprotuberance 32, wires 30 a and 30 b are not centered on downward member24. Instead these elements are offset relative to the center ofreservoir 40 such that when stirrer bar 18 is spun, these elements arenot in the center of the circular flow of sample where sample fluidmovement is slower, but rather in an offset location where fluid flow isfaster.

Cuvette subassembly 14 (and specifically upper body 46) further includesa downwardly sloping channel 54 (FIGS. 4, 5, 7 and 12) slopingdownwardly from an upper part of the cuvette subassembly to theapproximately the upper part of reservoir 40. Channel 54 thus allowsreagent(s) to be placed into a sample in reservoir 40 (usually bypipette) without the reagent placement substantially interferingmechanically with electrode 12.

Cuvette subassembly 14 also includes a pair of rearwardly projectingflanges 56 a, 56 b that extend rearward from arms 48 a, 48 b, forming apair of sidewalls on the cuvette that reduce the chance of hand contactwith the electrode subassembly 12 inside the cuvette subassembly 14. Inaddition, the lower ends of flanges 56 a and 56 b rest on theheater/stirrer assembly 16 (see, e.g., FIG. 10) and assist inpositioning the cuvette subassembly in the heater/stirrer assembly. Thepositioning and dimensions of flanges 56 a and 56 b each relative toback member 50 create a pair of openings 58 a and 58 b (see FIGS. 4-5and 10 that allow a pair of electrically conductive contacts (orbrushes) 60 a, 60 b (see FIGS. 9, 10 and 14) mounted on heater/stirrerassembly 16 to make electrical contact with pads 26 a and 26 b whenassembly 10 is mounted in assembly 16 as shown in FIGS. 10 and 14.

Assembly 10 preferably operates with stir bar 18 (FIGS. 8A and 12)adapted to be placed in the bottom of the cuvette reservoir 40, and freeto rotate under the influence of an outside source to create a flow ofsample in the well and between the two wires 30 a and 30 b of theelectrode subassembly when the electrode subassembly is mounted in thecuvette subassembly, the distances between bottom ends of the two wiresand the bottom of the lower reservoir being such that the stir bar canfit in that distance and still create such flow.

The heater/stirrer assembly 16 (FIGS. 9-12) includes a heater block 62that contains a well 63 (FIG. 12) that receives reservoir 40 whencuvette subassembly 14 is mounted in assembly 16. The heater block has aheating element (not illustrated) that can warm the block and thereservoir to an appropriate temperature (i.e. about 37° C.) for sampleanalysis.

Heater/stirrer assembly 16 (FIG. 12) also includes a stirrer motor 64that is operably connected via a motor shaft 66 to turn a magneticstirrer 68, which proximate enough to the bottom of reservoir 40 suchthat stirrer 68 can spin stir bar 18 in the bottom of reservoir 40 whenstirrer 68 is spun by motor 64. Thus, the sample in reservoir 40 can bemixed and circulated between wires 30 a and 30 b.

The upper portion of heater block 62 has a recess 70 (FIGS. 9 and 12)that generally conforms to the shape of cuvette subassembly 14 to holdthe cuvette subassembly 14 steady when in use and to allow good thermalcontact with the cuvette subassembly to allow the sample to be heated.

This invention allows for the monitoring of platelet aggregation in asmall sample size, using a low-cost disposable electrode assembly. Thiselectrode assembly consists of an electrode, a plastic cuvette and astir bar, which fits into a sample chamber. The sample chamber is heatedat 37° C. and the stir bar is typically spun at 1000 RPM but can be spunfrom 500 to 1200 RPM. The electrode has two fine palladium alloy wiresevenly spaced with both ends secured to Kapton. A small voltagedifference is applied through contacts 60, and ultimately across wires30 a and 30 b to measure the impedance of the sample. These wires areset apart about 0.18 to about 0.42 mm apart, which is close enough toallow a platelet aggregation plug to bridge the wires and produce astable result. The new electrode configuration allows the wires to becompletely submerged in a smaller sample volume. The position of thewires is important to accurate test results. The electrode wires shouldbe completely submerged within the sample (i.e. are completely coveredby the sample). When completely submerged the location of the wiresshould be where there is sufficient stirring with an even flow pattern,and where the wire electrodes do not interfere with the stir bar.

The cuvette subassembly can be a made from plastic so it can produced ata low cost using injection molding. The cuvette was designed with thefollowing features so that it fits this use:

-   -   Sample chamber is small enough so that electrode wires are        completely submerged in a smaller sample volume.    -   Two slots 38 a and 38 b for holding the electrode subassembly        securely in the position that thereby fixes the electrode wires        in the optimum position in the sample.    -   A flat-bottom reservoir 40 so that the stir bar 18 spins without        interference, which stirs the sample evenly and consistently.    -   Reinforcement member 34 at the top of the electrode        sub-assembly, located behind the contact pads 26 a, 26 b of the        electrode, which adds support to the substrate 20 when in        contact with contacts 60.    -   A channel 54 from the top of the cuvette subassembly to the        approximately mid-point, so that the pipette tip, used for        dispensing the reagent/agonist, can be inserted into the sample        without bumping against the electrode.

The cuvette is inserted into a sample chamber heated to 37° C. Thesample chamber consist of two parts, a socket, which has two springloaded contacts 60 that make electrical contact with the contact pads 26a, 26 b, and a heater block, which contains the heating element andtemperature sensors. Mounted below the heater block is a motor 64 andmagnet assembly 68 which when power is applied, spins at 1000 RPM. Themagnet mounted on the motor provides a magnetic force that spins thestir bar in the cuvette. There are cables and connectors mounted to theheater block assembly that connects components embedded in the heaterblock assembly to a printed circuit board (“PCB”; not shown).

On the PCB are conventional circuits which measure changes in resistancethat occur in the sample after the addition of an agonist. As is wellknown, the results of impedance aggregation tests are quantified by:

-   -   Ohms of aggregation at a given time in the test    -   Slope, or rate of the reaction, in ohms change per minute    -   Maximum extent of aggregation, in ohms.

The results can be recorded on a strip chart recorder or to a computerusing available software.

We claim:
 1. An electrode subassembly comprising: a. an electricallynon-conductive substrate with a top portion and a downwardly extendingmember; b. a pair of electrode contact pads separated from each otherand mounted on the top portion of the substrate, each contact pad havingan electrically conductive lead extending downwardly on the downwardlyextending member of the substrate, a conductive wire electricallyconnected to each lead, the conductive wire extending below anddownwardly away from the downwardly extending member, wherein theportions of the two wires that extend downwardly away are substantiallyparallel and are spaced from one another; wherein the electrodesubassembly comprise a part of an assembly for measuring plateletaggregation in a sample size of about 250 μL to 300 μL.
 2. The electrodesubassembly of claim 1, wherein the portions of the two wires that aresubstantially parallel are spaced from one another about 0.18 to about0.42 mm apart.
 3. The electrode subassembly of claim 2, wherein theportions of the two wires that are substantially parallel are from about1.9 to about 4.5 mm long.
 4. The electrode subassembly of claim 2,wherein the electrically conductive lead comprises a coating of anon-conductive material.
 5. The electrode subassembly of claim 2,wherein the downwardly extending member has a narrower protrudingportion where the upper end of the conductive wire is mounted.
 6. Theelectrode subassembly of claim 2, wherein the lower ends of the twowires comprise an electrically non-conductive spacer element fixing thetwo wires in parallel to one another.
 7. The electrode subassembly ofclaim 1, wherein the assembly for measuring platelet aggregationcomprises a cuvette subassembly.
 8. The electrode subassembly of claim7, wherein the electrode subassembly is adapted to fit into the cuvettesubassembly; wherein the top portion of the electrode subassembly is atthe top and the other end of the electrode subassembly is within thecuvette subassembly; wherein the electrode subassembly is relativelyfixed; and wherein there is a space between the end of the electrodesubassembly and an inner bottom portion of the cuvette subassembly. 9.The electrode subassembly of claim 2, wherein the assembly for measuringplatelet aggregation comprises a cuvette subassembly.
 10. The electrodesubassembly of claim 9, wherein the electrode subassembly is adapted tofit into the cuvette subassembly; wherein the top portion of theelectrode subassembly is at the top and the other end of the electrodesubassembly is within the cuvette subassembly; wherein the electrodesubassembly is relatively fixed; and wherein there is a space betweenthe end of the electrode subassembly and an inner bottom portion of thecuvette subassembly.